Engineering Disulphide-Free Autonomous Antibody VH Domains to modulate intracellular pathways

An attractive approach to target intracellular macromolecular interfaces is to design small high anity proteins. In this manuscript a stable, autonomous, human derived non-immunogenic, disulphide-free VH domain, has been engineered for intracellular expression studies. VH domains can be designed to possess a large dynamic repertoire of binders, as opposed to other scaffolds types that are highly rigid and possess fewer sites of random variation. Picomolar inhibitors were identied using phage display against the eIF4F complex, which is commonly hyper-activated in many cancers. These molecules were also shown to impair cellular proliferation and to reduce the expression of malignancy related proteins. Structural characterization elucidated that these VH domains bound eIF4E at the eIF4G interaction interface via a novel binding pose. Molecules able to mimic this pose and interfere with the eIF4F complex are potentially important for wide-ranging tumour therapy applications.


Introduction
Many intracellular macromolecular interfaces exist in human cancers that are highly desirable targets for the development of anti-cancer therapeutics, ranging from myc:max and b-catenin:TCF to KRAS:RAF 1 .
Some of these interactions present signi cant hurdles to small molecule development due to the underlying nature of their molecular surfaces. An attractive approach to circumvent these issues is to design and evolve small high a nity proteins ( approx. 60 to 100 amino acids in size) to target these interfaces, termed mini-proteins 2,3 . However, they are of special interest as they can easily be expressed within mammalian cells and engineered into transgenic mice, where they can be induced in a temporal and tissue speci c manner. In the absence of pharmacological speci c reagents they offer a precise biological tool for the interrogation of fundamental biology and target validation 4 .
Mini-proteins have several advantages over traditional knock-down and knock out strategies to validate candidate target proteins for therapeutic development, such as they more closely model the fundamental interactions of drugs with their targets and can also be designed to interact at the desired target sites 5 .
Additionally, mini-proteins can be used in animal models to study potential mechanisms of toxicity and establish attainable therapeutic thresholds in conjunction with disease models 5 . These types of studies will allow earlier identi cation of relevant clinical indications for pharmacological intervention enabling the streamlining of future therapeutic development. Many currently used miniproteins are limited as binding modules due to randomization of highly rigid structural motifs with limited sites of variation. 2 Variable domains of the human immunoglobulin heavy chain (VH domains) are ideal candidates for use as mini-proteins. Although of slightly larger size at approximately 120 amino acids, they possess three binding loops of variable length (CDR-H1, CDR-H2 and CDR-H3) that are naturally randomised to generate a wide repertoire of binders for antigen recognition by the immune system. Additionally, they should have negligible issues with immunogenicity issues in human therapy. However, the development of these scaffolds as single domain binding modalities has been restricted by poor stability, due to the loss of stabilizing interactions with the light-chain in the intact antibody 6,7 . Several groups have identi ed monomerically stable VH domains either by serendipitous discovery or by using phage-based evolution methods with the VH domain of trastuzumab (4D5) as a template. 6,8 Despite the progress in the generation of an autonomous, stable VH domain, there has been no success in generating a stable disulphide-free VH domain (DiF-VH), which can be used for intracellular applications.
In this manuscript we describe the use of CoFi 10 and Hot-CoFi 11 screening technologies to generate a stable, autonomous, disulphide-free VH domain (DiF-VH) based on the 4D5 antibody scaffold by directed evolution. This was then used to develop a novel VH domain (VH-S4) with picomolar a nity against the well-known oncogene eIF4E, which was suitable for intracellular expression and activity modulation studies. eIF4E forms part of the eIF4F complex, which performs a critical role in mediating cap-dependent protein synthesis 12 . The components (eIF4E, eIF4A and eIF4G) of which are also frequently overexpressed and/or mis-regulated in many tumours and often associated with poor prognosis in patients and chemoresistance [13][14][15] . The disulphide-free VH domain technology presented here offers a rapid and e cient pipeline to discover new binding poses for therapeutic lead development, as well as the identi cation of modalities that can be used to therapeutically model and validate these sites for drug development in mammalian and animal systems.

Results
Generation of a stable, disulphide-free, autonomous antibody variable domain for intracellular expression.
The 4D5 VH domain was chosen as the parental scaffold for stabilization, as its parental IgG trastuzumab is an approved FDA drug with a favourable immunogenic and stability pro le. 6 The 4D5 VH domain was fused to an N-terminal signal sequence for periplasmic expression in E. coli and a random mutagenesis library of the VH ORF was created. This was then used to initiate 2 rounds of selection using Hot-CoFi 11 ( Fig. 1A and 1B), resulting in the isolation of 53 stabilized VH domain variants. Among these, VH-36 and VH-36 were the most thermally stable, with T CAGG s of 73.4°C and 72.9°C, respectively, an increase of over 20°C in comparison to the parent WT clone (Fig. 1C). The thermostable VH-36 clone was then selected as the starting template for the evolution of a disulphide-free VH domain suitable for intracellular cell expression. Three residues in VH-36, C22 and C92, as well as the core residue A24, were randomised with all 20 amino acids to generate a library suitable for screening. The signalling peptide, stII, was also removed from the VH-36 expression construct (termed VH-36i) to ensure expression in the cytoplasm and allow screening for autonomously stable VH domains in a cellular reducing environment. This library was screened using the Hot-CoFi methodology and ve unique variants of VH-36i were identi ed ( Fig. 1A and 1C). These were then expressed and puri ed, and their thermal point of denaturation (T m ) determined by differential scanning uorimetry (DSF), with VH36i.1 demonstrating the highest thermal stability with a T m of 58°C. VH36i.1 possessed the mutations C22S, A24C and C92T ( Fig. 1A and 1C).
Optimization of a disulphide-free autonomous antibody variable domain for phage display.
The VH-36i.1 clone and its parent templates (VH-36 and VH-36i) all contained the mutation A100bP within the CDR-H3 loop. An undesirable feature with respect to target binding, as it restricts the conformational space open to randomized CDR-H3 loop regions and in turn decreases their probability of interacting successfully with target molecules. Therefore, the CDR-H3 region from position W95 to P100b was removed and replaced by the sequence SSSA, creating the new construct VH-37i, which had signi cantly reduced thermal stability as determined by DSF (T m =46°C). VH-37i, was then used to create a random mutagenesis library for CoFi screening 10 to compensate for this loss of protein stability. From this process 4 variants were identi ed with VH-37i.1 and VH-37.2 exhibiting the highest thermal stabilities (T m ), approximately 55.7°C for both ( Fig. 1A and 1C). Both clones contained a single mutation with VH-37i.1 bearing a A78V mutation in the core of the VH domain, whilst VH-37.2 contained the mutation G93V located near the CDR-H3 loop (Fig. 1D). Both mutations were combined to generate VH38i with a T m of approximately 57.3°C. To remove the nal cysteine in VH-38i at position 24 it was either mutated to the Ala residue found in the WT template, Ile, Val, Tyr, or Trp. The VH-38i variants containing either C24I or C24L substitutions were the most stable with improved T m s of 58.3°C and 58.3°C, respectively compared to the VH-38i template molecule (Fig. 1C).

VH Domain Phage Display library construction and Delineation of the VH-1A2 and VH-1C5 interaction sites on eIF4E
A library of VH domains were then displayed on the pIII protein of M13 phage, where the CDR1 and CDR2 of the VH-38i template were randomized conservatively, whilst CDR3 was randomized with sequences of different lengths and biased towards residues serine and tyrosine with approximately 20% frequency each. Phage display selection against puri ed human full-length eIF4E led to the identi cation of VH Domains VH-1A2 and VH-1C5 as the highest a nity binders with K d s of 115.2 ± 4.4 nM and 154.3 ± 70.3nM, respectively ( Fig. 2A, Table 1). The phage selected CDR3 loop sequences of the VH-1A2 and VH-1C5 domains shared little similarity to the interacting motifs of known eIF4E binding proteins such as eIF4G1 and the 4E-BP family (YXXXXLΦ, termed the canonical motif) (Fig. 2B). Competitive based uorescence anisotropy experiments were performed that mapped the binding of VH-1A2 and VH-1C5 to the eIF4G interaction site (Fig. 2B). Table 1 Binding and kinetics parameters of various VH domains against eIF4E. K d , k on and k off values were derived from SPR experiments using either single cycle or multi cycle injection experiments against eIF4E amine coupled to a CM5 sensorchip. Parameters were derived using a 1:1 site binding model. However, with regards to 4E-BP1 4ALA , binding parameters were initially determined using a 1:1 site binding model, which generated a poor t and physically irrelevant K on and K off values beyond the detection limit of the machine. This analysis was superseded with a 2-state analysis, which generated more meaningful kinetic parameters. The use of a 2-state model is supported by following reasons: 1) 4E-BP1 4ALA undergoes a disorder to order transition upon binding eIF4E and, 2) it interacts at eIF4E through 2 binding sites. Crystals were obtained for eIF4E complexed with m 7 GTP and VH-1C5 ( Figure S1), which revealed that VH-1C5 bound eIF4E at a position that overlaps with the eIF4G interaction site. The phage selected CDR3 loop (residues 111-120) of VH-1C5 forms a highly folded small domain structure that interacts directly with eIF4E, whose structure contrasts sharply with the 'L'-shaped conformers formed by 4E-BP1 and eIF4G derived linear peptides when in complex with eIF4E ( Fig. 2C and 2D) 16 . The random coil section (residues 101-107) of the CDR3 loop orientates the VH domain residue L105 into a position that mimics the interactions made by the conserved hydrophobic residue in the eIF4E interaction (residues M60 and L630 in 4E-BP1 and eIF4G1, respectively) (Fig. 2E). The random coil section of the VH domain also forms additional contacts with a hydrophobic region located on eIF4E unexploited by the canonical peptides 17,18 e.g., the side chain of A102 forms a hydrophobic contact with W73, whilst the sidechain of D104 packs against Y76 and L131. Y117, located on the helical segment (residues 108-117) of the CDR3 loop, forms hydrophobic packing interactions with L135 and a sidechain to sidechain hydrogen bond with E132 on the surface of eIF4E (Fig. 2E). These interactions replace the conserved electrostatic interaction mediated by R55 and R625 of the 4E-BP1 and eIF4G1 peptides with E132, respectively. F220, positioned on the short helical turn motif (residues 119-121) that precedes the loop re-joining the main body of the VH 1C5 fold, forms several hydrophobic contacts with the eIF4E residues L39, V69 and I138 and a backbone hydrogen bond interaction with the indole side chain of W73 on eIF4E (Fig. 2F). These interactions are replicated by the conserved Y and L residues of the eIF4E interaction motif (Y54/624 and L59/629 of 4E-BP1 and eIF4G1, respectively) with the backbone carbonyl of the L forming a hydrogen bond with W73.
Alanine Scanning Mutagenesis Reveals V H 1C5 Residues Distal to the Interaction Interface are Critical for Binding Alanine mutagenesis scanning of the CDR3 library insert (Fig. 3) was performed using yeast display.
These experiments revealed two classes of mutants that adversely affected the binding of VH 1C5 to eIF4E ( Fig. 3A and 3B). The rst class of mutations describe residues identi ed in the crystal structure that are in direct contact with eIF4E (K102, A103, D104, L105, T116, Y117 and F120). These results showed high correlation with residues that were identi ed through computational molecular dynamics simulations to signi cantly contribute energetically to the eIF4E:VH-1C5 complex interface ( Fig. 3C and Table S1). Interestingly, both alanine mutagenesis scanning and binding decomposition analysis identi ed that D104 forms unfavourable interactions at the eIF4E and VH-1C5 interface, and when replaced with alanine resulted in a VH-1C5 variant (VH-M4) that had an approximate 17-fold improvement in its a nity towards eif4E (Fig. 3D, Table 1) with a K d of 4.8 ± 1.7 nM. Inspection of the eIF4E: VH-1C5 structure revealed that the residues in proximity to D104 are primarily hydrophobic and that the D104A mutation allows for more favourable van der Waals interactions to occur.
The second class of mutations describe the alanine scanning replacement of residues V101, P110, V113, V114 and F122 that abolished eIF4E binding but were not located at the VH-1C5 interface. These residues form a distinct hydrophobic cluster that interact with several hydrophobic residues located on the β-sheet face of the immunoglobulin fold of the VH domain (V39, L47 and W49) (Fig. 3E). The sensitivity of VH-1C5 binding to eIF4E when this cluster is mutated suggests that these residues play a critical role in ensuring the correct folding of the CDR3 loop and spatial orientation of the contact residues. It also implies that the CDR3 loop forms a highly stable fold, a conclusion further supported by MD simulations that shows that the CDR3 loop structure of the unbound form deviates negligibly from the bound form ( Fig. 3F). A salt bridge also forms between residues R52 and D37 at the interface between the CDR3 loop and the VH domain fold, which orientates the R52 to form long range electrostatic interactions with the backbone carbonyls of S107 and S108 further rigidifying the CDR3 loop.
Evolution of an Ultra High A nity Mini-Protein Inhibitor of the eIF4E:4G interaction The improved VH-M4 binder (K d = 4.8 ± 1.7 nM) was used as a template sequence to generate a randomly mutagenized library for a nity maturation to identify improved binders against eIF4E using yeast display (Fig. 4A). Three rounds of kinetic selection were performed with increasing incubations times of 8 mins, 60 mins and 110 mins to increase the competitive pressure of unlabelled eIF4E against the VH domain library complexed to uorescent eIF4E (Fig. 4B). The nal round of kinetic selection resulted in the identi cation of only three eIF4E binders: VH-1C5 D104A/S108R/F120I , VH-1C5 D104A/S24G/F120I and VH-1C5 D104A/Y97C/F120I (Fig. 4C). The VH-1C5 D104A/S108R/F120I clone (termed VH-S4) dominated the nal round with a frequency > 90% and was selected for scale up and SPR analysis, which revealed that it interacted with a sub-nanomolar K d of 0.057 ± 0.004 nM (Fig. 4D, Table 1). The 2 substitutions identi ed in the VH-S4 sequence were then individually introduced into VH-1C5-M4 and their binding to eIF4E assessed. SPR analysis revealed that VH-1C5 D104A/S108R and VH-1C5 D104A/F120I (termed VH-S2) possessed K d s of 0.94 ± 0.04 nM and 0.64 ± 0.06 nM, respectively (Fig. 4, Table 1). Preliminary examination and MD simulations of the VH-1C5:eIF4E crystal structure with the identi ed mutations highlighted no substantial change in the energetics pro le of the interaction ( Figure S2) but did indicate that the F120I mutation most likely optimised the hydrophobic contacts between both proteins and allowed a more optimal engagement to occur. However, with regards to the S108R mutation, MD simulations showed only a transient interaction with R128 of eIF4E (Table S1).
The crystal structure of eIF4E: VH-1C5 D104A/S108R/F120I (VH-S4) complex was resolved, which enabled con rmation that the F120I mutation improved the complementarity of t between the two proteins ( Fig. 4E and S3). The elucidated complex structure also showed that the S108R mutation did not result in a direct interaction with the surface of eIF4E, but rather that it engaged a structured water network that forms an h-bond to the backbone carbonyl of R128 on eIF4E ( Fig. 4F and S3). Additionally, S108R formed a cation-π interaction with the sidechain of the neighbouring F112, further helping to stabilise the local fold of the CDR3 recognising eIF4E. The eIF4E: VH-1C5 D104A/S108R/F120I (VH-S4) crystal structure also revealed that the substitution of D104 with alanine results in a signi cant conformational change in Y76 upon the surface of eIF4E ( Fig. 4G and S3). This change is also associated with the subtle migration of several structure waters that enable the local H-bond network around the Y76 residue to be maintained.
The D104A mutation removes the unfavourable packing of the negatively charged D104 side chain from against Y76 and allows a more optimal packing arrangement to occur ( Fig. 4G and S3).

VH-S4 Disrupts eIF4F Complex Formation and Cap-Dependent Translation In Vitro
VH-S4 was expressed in HEK293 cells and shown to immune-precipitate endogenous eIF4E more strongly than the VH domain mutants with weaker a nities (VH-1C5, VH-M4 and VH-S2) for eIF4E (Fig. 5A). A VH-1C5 negative control (VH-1C5 SCRM ), where the residues in the CDR3 loop were scrambled, was also included that failed to pull-down eIF4E demonstrating the speci city of the VH domains for eIF4E (Fig. 5A). The ability of VH-S4 to interact with eIF4E was also compared to a constitutively active 4E-BP1 construct (termed 4E-BP1 4AlA ), where the phosphorylation sites (Thr37, Thr46, Ser65, Thr70) 19 responsible for modulating its binding with eIF4E were mutated to alanine. The K d of 4E-BP1 4ALA against eIF4E was determined to be 0.68 ± 0.13 nM approximately 10-fold weaker than the VH-S4 interaction with eIF4E, with a substantially weaker off-rate (Table 1 and Figure S4). Both constructs exhibited comparable levels of activity in immuno-precipitating eIF4E, whilst the 4E-BP1 4ALA negative control (termed 4E-BP1 YLM ) had negligible effects. VH-S4 was then shown to more e ciently disrupt the eIF4G-eIF4E interaction in cells than the weaker a nity VH domain variants (1C5, S2 and M4) using the NanoBit eIF4E:eIF4G 606-646 cell-based assay 20 (Fig. 5B). 4E-BP1 4ALA was also assessed in the NanoBIT assay and again was similar in activity to VH-S4. All the VH-domains except the control were then shown to inhibit cap-dependent translation in a bicistronic reporter assay, with the magnitude of cap-independent inhibition again correlating to the a nity of the eIF4E interacting VH domains (Fig. 5C). It is well established that disruption of the eIF4F complex prevents eIF4G mediated phosphorylation of eIF4E by Mnk1. 21 Therefore, in parallel, lysates were co-prepared from the cells used in the NanoBIT assay and the levels of phosphorylated eIF4E detected (Fig. 5C). eIF4E phosphorylation as expected closely followed the levels of eIF4F complex disruption measured in the NanoBit eIF4E:eIF4G 606-646 system. 4E-BP1 4ALA was also tested in both the bicistronic and eIF4E phosphorylation assays, where it speci cally decreased eIF4F complex formation and phosphorylation levels in a similar manner to the VH-S4 domain (Fig. 5C).
The effects of eIF4F complex disruption directly upon cap-dependent protein translation were monitored by measuring Cyclin D1 protein levels 22,23 . Increasing amounts of VH-M4 and VH-S4 expression plasmid were transfected into mammalian cells with a concomitant inhibitory effect on eIF4E phosphorylation and cyclin D1 protein levels (Fig. 5D), with the more tightly binding VH-S4 domain (K d = 60 pM) having a more profound effect. This effect of eIF4F disruption upon cyclin-D1 translation and eIF4E phosphorylation was also re ected by transfection experiments with 4E-BP1 4ALA (Fig. 5E and 5F). m 7 GTP mediated pull down experiments, using HEK293 transfected cells, veri ed that all the VH domains (S4, M4, S2 and 1C5) and 4E-BP1 4ALA , except for the controls, were able to competitively displace eIF4G from eIF4E and decrease the amount of endogenous eIF4F complex detected (Fig. 5E).
Vh-s4 Modulates Eif4f Mediated Signalling Pathways Speci cally eIF4E inhibition has been shown to reduce the expression of malignancy-related proteins (e.g., cyclin  Figure S5A) in comparison to mock control cells. Both proteins when induced for over 7 days also caused signi cant decreases in the cellular proliferation and viability (Figs. 6A, 6B, S5C and S5D). The most dramatic effects were seen in A375 cells, where both 4E-BP1 4ALA and VH-S4 decreased cellular proliferation and viability to the same extent ( Fig. 6A and 6B). In contrast, VH-S4 was less e cacious than 4E-BP1 4ALA in MD-MBA-231 cells, despite disrupting eIF4F complex formation to the same extent ( Figure S5A).
We next assessed the effects of both proteins on the expression of several proteins (Cyclin-D1, Mcl-1 and Bcl-xl) whose translation is regulated by the eIF4F complex in A375 cells (Fig. 6C). Both VH-S4 and 4E- and 72 hours (Fig. 6C). This result suggests that the down-regulation of Mcl-1 is insu cient to induce apoptosis in A375 cells and that the decrease in cellular proliferation measured with both VH-S4 and 4E-BP1 4ALA is principally driven by the reduction in Cyclin-D1 protein levels (Fig. 6C). Additionally, both VH-S4 and 4E-BP1 4ALA reduced the total protein expression of 4E-BP2 with negligible effects on 4E-BP1 levels.
Protein expression levels was reassessed post 72 hours doxycycline induction, which revealed that Mcl-1 was no longer signi cantly repressed but showed that 4E-BP1 levels were reduced with a concomitant decrease in its phosphorylated forms ( Figure S6A). This lack of sustained decrease in Mcl-1 levels offers further explanation of the lack of cellular apoptosis with eIF4F complex disruption. In parallel experiments, both VH-domains and 4E-BP1 4ALA inhibited global protein synthesis by approximately 50% as determined using puromycin pulse chase experiments ( Figure S6B). Results that correlated with the signi cant effects of eIF4F complex disruption upon cellular proliferation (Fig. 6A). We also veri ed the role of eIF4F in mediating STAT1 levels in IFN-γ treated A375 cells (Fig. 6D) 29 , where selective induction of VH-S4 or 4E-BP1 4ALA in IFN-γ treated cells reduced STAT1 protein levels.
The speci city of the VH-S4 domain was assessed by examining its effects on the pathways (AKT/mTORC and RAS/ERK) that regulate the eIF4F complex. Many of the chemical reagents used to study the biological function of the eIF4F complex modulate these pathways, and as a result elicit eIF4F independent effects e.g. PP242 30 , an ATP competitive inhibitor of mTORC1/2 that leads to dephosphorylation of both its downstream targets, 4E-BP1 and S6 kinase. A375 cells were treated with either PP242 or staurosporine (a non-selective kinase inhibitor) 31 and their effects on eIF4E, AKT, ERK and rS6 phosphorylation compared to disruption of the eIF4F complex by both 4E-BP1 4ALA and VH-S4 (Fig. 6C). Both miniproteins, caused dephosphorylation of eIF4E in a manner similar to PP242 through inhibition of eIF4G mediated MNK1 phosphorylation. 20 As expected, PP242 reduced 4E-BP1 phosphorylation to mediate its known effects on eIF4F complex disruption 30 . However, PP242 unlike the miniproteins also reduced phosphorylation of rS6 and AKT phosphorylation though its effects on mTORC12 (Fig. 6C) 30,32 . A375 cells treated with 0.2 µM of the broad kinase inhibitor staurosporine resulted only in the dephosphorylation of S6 kinase. Neither the miniproteins nor the small molecules tested affected ERK phosphorylation.
The effects of small molecules upon the eIF4F complex were extended to a wider set of compounds (including the eIF4E inhibitor, 4EGI-1 and the Mnk Kinase inhibitor, CPG-57380) and assessed at 72 hours (Fig. 6E). The effects of PP242 and staurosporine on protein expression levels of Mcl-1 at 24 hours ( Fig. 6C) were alleviated by 72 hours (Fig. 6E), whilst maintained with respect to cyclin-D1, 4E-BP1 and 4E-BP2. CPG-57380 as expected resulted in dephosphorylation of eIF4E at 72 hours ( Fig. 6E) with negligible effects on cyclin-D1 or 4E-BP1/2 protein levels. Additionally, PP242 and staurosporine induced apoptosis at 72 hours as indicated by PARP cleavage (Fig. 6E and Figure S6C)) in contrast to the miniproteins demonstrating that speci c inhibition of the eIF4E:4G interface does not lead to apoptosis and that this is the result of other modes of action by either small molecule. Interestingly 4EGI-1, a molecule that allosterically disrupts binding of eIF4G to eIF4G, also elicits PARP cleavage and promotes eIF4E phosphorylation in contrast to VH-S4 and 4E-BP1 4ALA .

Discussion
The rapid generation of high a nity mini-proteins against protein-protein interactions intractable to small molecule development represents a valuable methodology for target validation and therapeutic modelling. We have developed a repertoire of VH domain-based mini-proteins that sample a wide range of K d s (picomolar to micromolar), which all disrupt the eIF4F complex though a novel binding pose and that are all amenable to cellular expression. A unique feature of the evolved eIF4E interacting VH domains is that the randomised CDR3 loop forms a well-de ned domain type structure. In contrast to PPIs such as Mdm2:p53 33 and Bcl-2:Bax 34 , the eIF4E:4G 16 interaction interface is relatively planar with no distinguishing clefts and is much more structurally similar to PPIs such as the interface found in the Notch transcription complex between ICN:CSL and MAML and β-catenin:TCF. 35 The eIF4E interacting VH domains have in effect evolved a module that is optimized for recognizing planar PPI surfaces, and with randomization of the appropriate residues that are non-critical to the stabilizing core residues, should serve as an ideal template for building libraries to explore these molecular surfaces further. Additionally, no clinically approved drugs exist that target the eIF4E:4G interface directly, despite the description of several antagonistic small molecules in the literature that albeit interact weakly with eIF4E and have little cellular e cacy. 12 The VH-S4 and VH-1C5 structures offer two possible templates for future lead molecule development: 1) the CDR3 loop could be used as a template to drive forward a macrocyclic discovery strategy and 2) the critical residues involved in binding eIF4E could be used as a basis for small molecule design.
The applicability of VH domains for cellular pathway modulation and therapeutic modelling were demonstrated by comparing their mode of action to several small molecules and a phosphorylation de cient mutant of 4E-BP1 (4E-BP1 4ALA ) known to modulate the eIF4F complex. Both 4E-BP1 4ALA and VH-S4 exhibited similar potencies to each other in terms of their bioactivity, which is surprising as VH-S4 interacts more strongly with eIF4E (Table 1). Both proteins also retarded cellular proliferation to a similar extent in A375 cells and exhibited a similar pro le in down-regulation expression of Mcl-1, cyclin D1 and 4E-BP1/2 protein levels at 24 and 72 hours. Interestingly, 4E-BP1 4ALA was more effective in reducing cellular proliferation of MB-MDA-231 cells despite similar e cacies to VH-S4 in terms of eIF4F complex disruption ( Figure S5). These results suggest that other mechanisms apart from displacement of eIF4G are putatively playing a role in the effects of 4E-BP1 4ALA mediated inhibition. Such effects could be post translational modi cations apart from phosphorylation (e.g., ubiquitination of eIF4E:4E-BP1 4ALA leading to increased degradation) or additional inhibition by endogenous 4E-BP1 through unappreciated feedback loops. This demonstrates that VH domains that are inert to cellular regulation pathways can be used to decipher their effects on naturally occurring repressors e.g., 4E-BP1, provided they interact at the same binding site.
Furthermore, the VH-S4 domain was compared to several well-known compounds that have been used to study the biological role of the eIF4F complex. For example, inhibitors of mTORC1, the multi-subunit protein responsible for phosphorylating 4E-BP1 and controlling its ability to inhibit eIF4E, is also responsible for regulating the activity of other target proteins and their downstream pathways, such as the rS6 kinase and the elongation step of protein synthesis. 36,37 Here we could demonstrate that VH-S4 exquisitely disrupts eIF4F complex disruption in the absence of effects on rS6 kinase phosphorylation (including 4EBP1 also) and is thus a more appropriate tool for understanding the biological impact of eIF4E. Additionally, we also compared it to the small molecule inhibitor of the eIF4E:4G interaction, 4EGI-1 38 , where in stark contrast to the VH-S4 domain the induction of apoptosis was seen. A result shared with the in mTORC1 inhibitor that was used (PP242). These results further demonstrate how highly speci c mini-proteins such as the VH-S4 can be used to re ne our understanding of the impact of small molecule inhibition on cellular phenotypes, and to delineate whether desirable outcomes such as apoptosis are the result of direct on target engagement or indirect off-target effects. Tools that could be invaluable for therapeutic design and research.
In summary, we have generated a unique disulphide-free VH domain scaffold optimized for intracellular folding and function and demonstrated how to generate picomolar-a nity VH domains to target and inhibit therapeutically attractive intracellular PPI interfaces through a unique binding mode. This unique binding mode can be used to inspire the new design of novel modalities and small molecules. VH domain display technology therefore represents a powerful valuable tool to discover new modes of inhibition and mini-proteins for exploratory biology and therapeutic modelling against macromolecule interfaces. We envision that in the future combined with new and novel alternate delivery methods e.g., RNA, toxin, and cell-penetrating peptide mediated delivery methods, that mini-proteins could also be used to target intracellular targets of interest and constitute potential therapeutics.